Background & objectives: It is imperative to know the aetiology of acute encephalitis syndrome (AES) for patient management and policy making. The present study was carried out to determine the prevalence of common aetiological agents of AES in Uttar Pradesh (UP) state of India.
Methods: Serum and/or CSF samples were collected from AES patients admitted at Gandhi Memorial and Associated Hospital, King George's Medical University, Lucknow, a tertiary care centre, UP during 2014–16. Cerebrospinal fluid (CSF) and serum samples from cases were tested for IgM antibodies against Japanese encephalitis virus (anti-JEV), and dengue virus (anti-DENV) by ELISA; and for enterovirus, herpes simplex virus (HSV) and varicella zoster virus (VZV) by real-time PCR. Serum samples of cases having sufficient CSF volume, were also tested for anti-scrub typhus IgM antibodies and for Neisseria meningitides, Streptococcus pneumoniae and Haemophilus influenzae.
Results: JEV and DENV (8% each) were the most common identified aetiology from the 4092 enrolled patients. Enterovirus, HSV and VZV, each were detected in <1% AES cases. Co-positivity occurred in 48 cases. Scrub typhus (31.8%) was the most common aetiology detected. Haemophilus influenzae and S. pneumoniae were detected in 0.97 and 0.94% cases, respectively, however, N. meningitides was not detected in any of the cases. About 40% of the JEV/DENV positive AES cases were adults. The gap between the total number of AES cases and those with JEV/ DENV infection increased during monsoon and post-monsoon seasons.
Interpretation & conclusion: Scrub typhus, JEV and DENV are the main aetiological agents of AES in UP. DENV and JEV can no longer be considered paediatric diseases. The prevalence of non-JEV/DENV aetiology of AES increases in the monsoon and post-monsoon seasons.

Background & objectives: Vector-borne pathogen surveillance programmes typically rely on the collection of large numbers of potential vectors followed by screening protocols focused on detecting pathogens in the arthropods. These processes are laborious, time consuming, expensive, and require screening of large numbers of samples. To streamline the surveillance process, increase sample throughput, and improve cost-effectiveness, a method to detect dengue virus and malaria parasites (Plasmodium falciparum) by leveraging the sugar-feeding behaviour of mosquitoes and their habit of expectorating infectious agents in their saliva during feeding was investigated in this study.
Methods: Dengue virus 2 (DENV-2) infected female Aedes aegypti mosquitoes and P. falciparum infected female Anopheles stephensi mosquitoes were allowed to feed on honey coated Flinders Technical Associates —FTA® cards dyed with blue food colouring. The feeding resulted in deposition of saliva containing either DENV-2 particles or P. falciparum sporozoites onto the FTA card. Nucleic acid was extracted from each card and the appropriate real-time PCR (qPCR) assay was run to detect the pathogen of interest.
Results: As little as one plaque forming unit (PFU) of DENV-2 and as few as 60 P. falciparum parasites deposited on FTA cards from infected mosquitoes were detected via qPCR. Hence, their use to collect mosquito saliva for pathogen detection is a relevant technique for vector surveillance.
Interpretation & conclusion: This study provides laboratory confirmation that FTA cards can be used to capture and stabilize expectorated DENV-2 particles and P. falciparum sporozoites from infectious, sugar-feeding mosquitoes in very low numbers. Thus, the FTA card-based mosquito saliva capture method offers promise to overcome current limitations and revolutionize traditional mosquito-based pathogen surveillance programmes. Field testing and further method development are required to optimize this strategy.

Background & objectives:Anopheles sinensis Wiedemann is a major vector of malaria and is among the dominant species in Shandong province of China. Knowledge of the blood-feeding patterns of mosquitoes is crucial for elimination of malaria vectors. However, little information is available on the blood-feeding behaviour of An. sinensis mosquitoes in Shandong province. This study was carried out to compare the blood-feeding behaviour of An. sinensis in malaria-endemic areas of Shandong province China.
Methods: Adult Anopheles mosquitoes were collected from three malaria-endemic areas (Jimo, Yinan and Shanxian), during the peak months of mosquito population (August and September) from 2014 to 2015. Indoor-resting mosquitoes and outdoor-resting blood-fed females were sampled in the morning hours (0600 to 0900 hrs) from 10 randomly selected houses using pyrethrum spray catch method, and sweeping with an insect net. ELISA was used for the identification of blood meal. The blood meal of each mosquito was tested against antisera specific to human, pig, dog, cow, goat, horse (mule) and fowl.
Results: At all indoor study locations of Jimo, Yinan and Shanxian, 59.4, 68.1 and 98.8% blood-engorged female An. sinensis collected from cattle sheds fed almost exclusively on bovines, respectively. For outdoor locations, at Jimo site, 27.27 and 49.55% An. sinensis fed on cattle and pigs; at Yinan, 30.42% fed on cattle and 36.88% fed both on cattle and goats, while no pig antibodies were detected. At Shanxian, percent of An. sinensis that fed on cattle, pigs and cattle-goat was 20.72, 27.62 and 21.78%, respectively.
Interpretation & conclusion: The analysis of An. sinensis blood meals in all the three studied areas from human houses, cattle sheds, pig sheds and mixed dwellings revealed that An. sinensis prefers cattle hosts, and can feed on other available animal hosts if the cattle hosts are absent, and the mosquitoes readily feed on humans when domestic animals (cattle and pigs) are not nearby for feeding. The analysis of blood meal revealed that An. sinensis follow opportunistic feeding in Shandong province, China.

Background & objectives: Rift Valley fever (RVF) is a zoonotic vector-borne disease that primarily affects domestic animals but can also infect humans. The purpose of the present study was to investigate the presence of antibodies against RVF virus (RVFV) in ruminants, viz. cattle, sheep, and goats in Kurdistan Province of western Iran.
Methods: Blood samples were collected from 288 ruminants (118 cattle, 142 sheep and 28 goats) of both sexes, under age groups ≤1, 1–3, 3–5 and ≥5 yr, from January 2016 to December 2016. Clinical symptoms and history of abortion were recorded. The presence of RVFV-specific antibodies was investigated by using ELISA (competitive) and indirect immunofluorescence assay (IIFA) after separation of serum.
Results: The results of two tests were positive for five (1.74%) of total 288 animals which included two cattle of 118 (1.7%), and three sheep of 142 (2.11%). The results of IIFA were correlated with the ELISA results. All animals were clinically normal. No significant relationship between the RVFV infection rate and the variable considered, i.e. season, animal’s age or sex, and the species of the animal (p ≥ 0.05), although there were four seropositive animals in the age group 1–3 and five seropositive animals in the spring season.
Interpretation & conclusion: The results of the study revealed the presence of low-level RVFV circulation among the ruminants of Kurdistan Province in Iran indicating that they are at risk of exposure to the virus during their lifetime. Since the present study was the first serological study on RVF in Iran with positive results, further studies are suggested including other areas of Iran.

Background & objectives: Cyprus is located in the eastern part of the Mediterranean Region where leishmaniasis is endemic. The primary objective of this study was to investigate human visceral leishmaniasis (VL) in the northern region of Cyprus where presence of canine leishmaniasis (CanL) and sandflies has been documented in earlier studies. The secondary objective was to assess the association of leishmaniasis with demographic and epidemiological variables.
Methods: Intravenous blood samples were collected from 249 volunteers in Kyrenia district (located in the northern coastal region of Cyprus). Whole blood samples were tested for DNA of Leishmania spp by polymerase chain reaction (PCR), while serum samples were analyzed using direct agglutination test (DAT) and rK39 test. For evaluation of possible risk factors, a questionnaire was applied to the participants.
Results: Only three (1.2%) of 249 participants were found seropositive by DAT (n = 2) or rK39 test (n = 1). The remaining samples were negative in serology, and no PCR positivity was detected in any of the 249 participants. Seven individuals, including the seropositive cases, had a history of cutaneous leishmaniasis (CL). Seropositivity and CL were not significantly related with gender (M/F: 40.2/59.8%), age [Mean: 42.85 ± 17.45, Median: 40 (7–86)], occupation (Indoor/Outdoor: 84.7/12.9%), dog ownership (52.6%), and CanL history (5.3%). However, a statistical association was found between seropositivity and past CL infection. Also, a significant relation was observed between participants living in peripheral area (63.1%) and CL infection. Furthermore, leishmaniasis awareness (28.1%) among the study population was statistically correlated with past CL infection and dog ownership.
Interpretation & conclusion: This study demonstrates the presence of leishmaniasis and highlight the need for implementation of efficient control measures on the northern coast of Cyprus.

Background & objectives: Crimean-Congo haemorrhagic fever virus (CCHFV) causes severe disease with fatality rate of 30%. The virus is transmitted to humans through the bite of an infected tick, direct contact with the products of infected livestock as well as nosocomially. The disease occurs sporadically throughout many of African, Asian and European countries. Different species of ticks serve either as vector or reservoir for CCHFV. This study was aimed to determine the prevalence of CCHFV in hard ticks (Ixodidae) in the Golestan Province of Iran.
Methods: A molecular survey was conducted on hard ticks (Ixodidae) isolated from six counties in Golestan Province, north of Iran during 2014–15. The ticks were identified using morphological characteristics and presence of CCHFV RNA was detected using RT-PCR.
Results: Data revealed the presence of CCHFV in 5.3% of the ticks selected for screening. The infected ticks belonged to Hyalomma dromedarii, Hy. anatolicum, Hy. marginatum and Rhipicephalus sanguineus species.
Interpretation & conclusion: The study demonstrated that Hyalomma ticks are the main vectors of CCHFV in Golestan Province. Thus, preventive strategies such as using acaricides and repellents in order to avoid contact with Hyalomma ticks are proposed.

Background & objectives: Entomological investigations were carried out in highly malarious villages under Ujina PHC of District Nuh (Haryana state) which is an epidemic prone area in northwestern region of India. The study was aimed to have an in-depth understanding of the entomological parameters influencing malaria transmission in the study area.
Methods: The seasonal prevalence and biological attributes of vector mosquitoes were investigated during 2015 and 2016. Indoor resting vector mosquitoes were collected from human dwellings/cattle sheds and morphologically identified. Anopheles culicifacies were categorized to sibling species by species-specific inversions in polytene chromosomes and An. stephensi to ecological races on the basis of ridge number on egg float. The blood meal source analysis and incrimination studies of vectors were done by counter-current immunoelectrophoresis and enzyme-linked immunosorbent assay, respectively. Insecticide susceptibility test on vectors was performed as per WHO guidelines.
Results: Seasonal abundance of An. culicifacies and An. stephensi in the study area showed variation; the peak densities of both the vectors were observed during monsoon months which correlated well with the average monthly rainfall data. Though both vectors were found to be primarily zoophagic, the human blood index of An. culicifacies (HBI = 0.17) was significantly higher than that of An. stephensi (HBI= 0.02). Analysis of sibling species composition of An. culicifacies population showed that it comprised almost of sibling species A (>98%) which is an established malaria vector. Anopheles culicifacies was incriminated for Plasmodium vivax and P. falciparum circumsporozoite (CS) antigen during monsoon months in 2015 and 2016. Assessment of insecticide susceptibility status of malaria vectors against 0.5% deltamethrin revealed that An. culicifacies is more susceptible (95% mortality) than An. stephensi (85% mortality).
Interpretation & conclusion: The results suggest that An. culicifacies (species A) is playing a major role in malaria transmission in the study area and is almost susceptible to deltamethrin. Timely two rounds of indoor residual spray of synthetic pyrethroid with proper dosage and good coverage would be helpful in reducing vector population and consequently the malaria incidence. In addition, personal protection measures by the community would supplement the major intervention tool (IRS) in decreasing the man-vector contact.

Background & objectives: Q fever caused by Coxiella burnetii is a zoonotic infection that spreads to human beings from animals. This study was aimed to demographically examine the C. burnetii seroprevalence in the people living in villages where Crimean-Congo haemorrhagic fever virus (CCHFV) is endemic, in terms of various risk factors such as tick bites, tick contact, and occupational groups.
Methods: A total of 440 serum samples from those living in rural areas of Sivas and Tokat regions in Turkey were included in the study as a risk group; 387of them were serologically CCHFV positive (as confirmed in our previous research). Serums of the control group composed of 110 people living in urban areas. In all serum samples, IgG antibodies of C. burnetii against phase-I and phase-II antigens were diagnosed using the ELISA method.
Results:Coxiella burnetii seropositivity was detected in 19.09% of those living in rural areas and 4.55% of those living in urban areas (p < 0.001, OR = 4.96). In terms of their approach to the ticks, no statistical difference was observed between the risk groups in the chi-square test (p = 0.787). However, according to univariate analysis, the absorbance means of antibodies reactive to C. burnetii was statistically higher for the rural people who have made contact with ticks than those who have not (p = 0.017). No seroepidemiological relation was found between CCHFV and C. burnetii serology (p = 0.787), and the rate of co-seropositivity between them was 5.43% (21/387).
Interpretation & conclusion: The findings of the study showed that C. burnetii infection is epidemic especially in the people living in rural areas. Contact with ticks in various ways might have resulted in the increased risk of C. burnetii infection in the study. Personal protective measures against tick bites may be important for reducing Q fever risk as in other tick-borne infectious disease.

Background & objectives: The nature of the rickettsial antigens and the immune response generated by them, have been the subject of exhaustive research so that a suitable vaccine can be developed. Till date evaluations of Rickettsia rickettsii antigens that induce both humoral and cellular responses in animal models have only shown partial protection and short-term immunological memory. This study was aimed to evaluate the immune response induced by DNA plasmids generated from the OmpA and OmpB genes of R. rickettsii in peripheral blood mononuclear cells of rickettsial (sensitized) patients compared to healthy subjects.
Methods: Plasmids OmpA-49, OmpB-15 and OmpB-24 were generated in the pVAX vector. Macrophages derived from the THP-1 cell line were transfected in vitro with the plasmids and were co-cultured with T-lymphocytes from sensitized subjects and healthy subjects to evaluate cell proliferation and cytokine production.
Results: The OmpB-24 plasmid induced proliferative response in human lymphocytes, with production of IL-2, IFN-γ, IL-12p70, IL-6 and TNF-α, likely due to the presence of conserved epitopes among R. rickettsii, R. typhi and R. felis (differing from 1 to 3 amino acids) during the construction of the plasmids.
Interpretation & conclusion: DNA sequences of rickettsial epitopes can be cloned into the pVAX vector. Constructed plasmids can generate a proliferative response and produce cytokines in vitro, in co-culture of transfected macrophages with sensitized human lymphocytes. Plasmid OmpB-24 proved to be the most immunogenic with respect to plasmids OmpA-49 and OmpB-15.

Aedes vittatus (Bigot) mosquito is a voracious biter of humans and has a geographical distribution throughout tropical Asia, Africa and the Mediterranean region of Europe. It is predominantly a rock-hole breeder, though it can breed in diverse macro- and micro-habitats. The mosquito plays an important role in the maintenance and transmission of yellow fever (YFV), dengue (DENV), chikungunya (CHIKV) and Zika (ZIKV) viruses. It has been implicated as an important vector of YFV in several African countries as evidenced by repeated virus isolations from the mosquito and its potential to transmit the virus experimentally. Similarly, DENV-2 has been isolated from wild caught Ae. vittatus mosquitoes in Senegal, Africa which has been shown to circulate the virus in sylvatic populations without causing human infection. Experimental studies have shown replication of the virus at a low scale in naturally infected mosquitoes while high rate of infection and dissemination have been reported in parenterally infected mosquitoes. Natural isolation of ZIKV has been reported from Senegal and Cote d’Ivoire from these mosquitoes. They were found highly competent to transmit the virus experimentally and the transmission rate is at par with Ae. leuteocephalus, the primary vector of ZIKV. A few CHIKV isolations have also been reported from the mosquitoes in Senegal and other countries in Africa. Experimental studies have demonstrated high susceptibility, early dissemination and efficient transmission of CHIKV by Ae. vittatus mosquitoes. The mosquitoes with their high susceptibility and competence to transmit important viruses, viz. YFV, DENV, CHIKV and ZIKV pose a major threat to public health due to their abundance and anthropophilic behaviour.